Constructs comprising fatty acids

Abstract

The present invention relates to a construct comprising a C13 to C27 fatty acid non-covalently bound to a hydrophobic region of a carrier particle, methods of manufacture, and uses thereof.

Claims

1. A method for treating a disorder selected from the group consisting of inflammatory bowel disease (IBD), ulcerative colitis, and Crohn's disease, the method comprising: administering to a subject in need thereof an effective amount of a construct obtained by a method comprising: admixing a fatty acid composition and a non-biological and non-biodegradable carrier particle under agitation to provide a homogeneous suspension, wherein the fatty acid composition comprises a C13 to C27 fatty acid, and wherein the non-biological and non-biodegradable carrier particle comprises a hydrophobic region; admixing water with said homogeneous suspension under agitation to facilitate non-covalent binding of an aliphatic chain of the C13 to C27 fatty acid to the hydrophobic region of the non-biological and non-biodegradable carrier particle; and removing excess water and excess fatty acid thereby treating said disorder.

2. The method of claim 1, wherein the C13 to C27 fatty acid comprises a C14 to C26 fatty acid or a C17 to C19 fatty acid selected from a linoleic acid, a palmitic acid, a stearic acid, an oleic acid, and/or a linolenic acid; the non-biological and non-biodegradable carrier particle is a silica particle having an average diameter of about 3.5 μm; and the construct is administered to the subject as part of a foodstuff.

3. The method of claim 1, wherein the C13 to C27 fatty acid comprises a C14 to C26 fatty acid or a C17 to C19 fatty acid.

4. The method of claim 1, wherein the C13 to C27 fatty acid comprises a linoleic acid, a palmitic acid, a stearic acid, an oleic acid, and/or a linolenic acid.

5. The method of claim 1, wherein the non-biological and non-biodegradable carrier particle is a silica particle, a kaolin particle, or a vermiculite particle.

6. The method of claim 1, wherein the non-biological and non-biodegradable carrier particle has an average diameter of about 1.0 μm to about 10.0 μm.

7. The method of claim 1, wherein the construct is administered to the subject as part of a foodstuff.

8. The method of claim 7, wherein the construct does not cross a mucosal layer of a gastrointestinal tract of the subject.

9. The method of claim 1, wherein the subject is a human, an equine, a canine, a bovine, a porcine, an ovine, a caprine, a feline, a chicken, a rodent, a fish, or a camelid.

10. The method of claim 1, wherein the treating modulates the subject's microbiota.

11. The method of claim 1, wherein the treating modulates the subject's buccal flora.

12. The method of claim 1, wherein the treating alters a composition of the subject's microbiota.

13. The method of claim 1, wherein the treating increases permeability of the subject's gut to bacterial vesicles or microvesicles.

14. The method of claim 1, wherein the treating increases permeability of the subject's gut to food, microbiota or components thereof.

15. The method of claim 1, wherein the treating modulates the subject's hypothalamus-pituitary-adrenal (HPA) axis.

16. The method of claim 1, wherein the disorder is IBD.

17. The method of claim 1, wherein the disorder is ulcerative colitis.

18. The method of claim 1, wherein the disorder is Crohn's disease.

19. The method of claim 1, wherein all excess water and all excess fatty acid is removed.

20. A method for manufacturing a construct for oral administration comprising: admixing a fatty acid composition and a non-biological and non-biodegradable carrier particle under agitation to provide a homogeneous suspension, wherein the fatty acid composition comprises a C13 to C27 fatty acid, and wherein the non-biological and non-biodegradable carrier particle comprises a hydrophobic region; admixing water with said homogeneous suspension under agitation to facilitate non-covalent binding of an aliphatic chain of the C13 to C27 fatty acid to the hydrophobic region of the non-biological and non-biodegradable carrier particle; and removing excess water and excess fatty acid.

21. The method of claim 20, wherein all excess water and all excess fatty acid is removed.

22. A method for treating a disorder, the method comprising: administering to a subject a construct, the construct obtained by a method comprising: admixing a fatty acid composition and a non-biological and non-biodegradable carrier particle under agitation to provide a homogeneous suspension, wherein the fatty acid composition comprises a C13 to C27 fatty acid, and wherein the non-biological and non-biodegradable carrier particle comprises a hydrophobic region; admixing water with said homogeneous suspension under agitation to facilitate non-covalent binding of an aliphatic chain of the C13 to C27 fatty acid to the hydrophobic region of the non-biological and non-biodegradable carrier particle; and removing excess water and excess fatty acid; thereby treating the disorder.

23. The method of claim 22, wherein all excess water and all excess fatty acid is removed.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.

(2) FIG. 1 shows a scanning electron microscopy (SEM) image of the construct of the invention.

(3) FIG. 2 shows the expression of β-catenin (red) and the position of the nucleus (blue—centre of cells) in (A) control untreated cells (B) Wnt treated (C) Wnt+Construct. The data show an increase in staining for β-catenin in (C) when compared to (A) and (B). Cf. the increase in signal in (C) (301=whole cell, 302=nucleus) when compared to the signal in (B) (201=whole cell, 202=nucleus), and (A) (101=whole cell, 102=nucleus).

EXAMPLES

Example 1

(4) Preparation of the Construct

(5) 54 g of pharmaceutical grade silica particles (average diameter ˜3.5 μm) were mixed with 1 L of rapeseed oil at room temperature and whisked to avoid clumping of the silica particles and obtain an evenly dispersed homogeneous mixture.

(6) Water was slowly added to a final volume of 2 L (1:1 v/v oil to water ratio). The admixture was left overnight to separate into three layers: a silica particle layer containing constructs comprising fatty acid non-covalently bound to the silica particles; an oil layer; and a water layer. The oil and water layers were removed and the oil recycled. The silica particle layer containing the construct of the invention was baked in an oven at 110° C. for 6 hours and blended to a fine powder.

Example 2

(7) Scanning Electron Microscopy of the Construct

(8) The construct was applied to conductive carbon tape on an aluminium SEM stub and then coated to 0.8 nm thickness using an Agar High Resolution Sputter Coater using a platinum/palladium target and imaged with a Hitachi S-4700 FESEM scanning electron microscope. FIG. 1 shows that the construct forms uniform particles.

Example 3

(9) Constituents of the Construct

(10) 10 mg of construct was added to 100 ml of sterile water, and a culture of active Schistosome cercaria cells added. The cells were observed under a microscope for 1 hour during which the majority of the Schistosome cercaria lost their flagellae. This was in stark contrast to a control (no construct added) in which after the same time period the majority of Schistosome cercaria cells retained their flagellae.

(11) Deflagellation occurs when motile cercaria come in contact with the linoleic acid on human skin and is a precursor to the deflagellate cercaria burrowing through the skin into the body of the host in order to complete their life cycle.

(12) The results of this experiment validate the presence of fatty acids bound to the silica particles, and in particular demonstrate the presence of linoleic acid bound to the silica particles.

Example 4

(13) Intracellular Release and Migration of β-Catenin

(14) Cells of a prostate cancer cell line (PC3) were exposed to the construct (suspended in phosphate buffered saline at 0.01 g/ml) or a SiO.sub.2 control for 25 minutes with or without Wnt (100 ng/ml) in a chamber slide. The cells were washed after treatment and fixed in 4% formalin for 30 minutes at 4° C. Cells were washed 3 times with ice-cold PBS and stored in 0.5 ml of PBS at 4° C. for immunostaining.

(15) For immunostaining, cells were washed twice for 5 minutes in a Tris base buffer (TB) containing 20 mM Tris, pH 7.4, 0.9% NaCl, 0.1% Tween 20. Cells were then incubated in 20% normal goat serum/TB for 30 minutes at room temperature, the chambers were drained, and cells were incubated with mouse anti-β-catenin (ab22656, Abcam) overnight at 4° C. Following two washes in TB, cells were incubated in goat anti-mouse peroxidase Fab (Abcam). Following a further two 5-minute washes in TBST, cells were incubated with tyramide Cy3 for 10 minutes at room temperature. Cells were washed twice for 5 minutes in TBST before nuclear counterstaining with DAPI. After a final two 5-minute washes, a coverslip was placed on the slides.

(16) Fluorescence immunocytochemical staining for β-catenin expression in cell lines was visualized using a confocal inverted microscope using a 20×0.7 numerical aperture or 40×1.0 numerical aperture dry objective. DAPI (nuclear counterstain) was excited at 405 nm with an emission of 415-500 nm; Cy3 (β-catenin-labeled) signal was excited at 561 nm with an emission spectrum between 570-670 nm. Laser power, gain, and off-set settings for the photomultiplier tubes were kept similar for comparable samples.

(17) The results show the expression of β-catenin (red) and the position of the nucleus (blue) in (A) control untreated cells (B) Wnt treated (C) Wnt+Construct. The data demonstrate an increase in the translocation of β-catenin in the cells treated with the construct of the invention.

(18) In conclusion, these data indicate that the constructs of the invention are pharmaceutically active. Moreover, the Wnt signalling pathway has been implicated in behaviour, with dysfunctional Wnt signalling aberrantly regulating new neurone development and cognitive function. Indeed many conventional mood stabilisers mediate their effect via modulation of Wnt signalling. Specifically, it has been found that rats treated with the mood stabilisers, lithium and valproate, show increased levels of β-catenin, which is indicative of an activation of the Wnt pathway. Likewise, a number of antipsychotics (e.g. Haloperidol, and Risperidone) and antidepressants (e.g. Fluoxetine, Fluvoxamine, and Venlafaxine) have been determined to increase Wnt/β-catenin signalling. Therefore the benefits of the construct in improving behaviour may (at least in part) be achieved by modulating Wnt/β-catenin signalling.

Example 5

(19) c-Fos Expression

(20) Dose of Construct

(21) Animals are dosed based on weight. The concentration of construct used is 33.33 mg/kg (a 750 kg horse receives a dose of 25 g), e.g. for a 300 g rat: 0.3 kg×33.33 mg=9.999 mg (10 mg). The construct is ground into a fine powder and solubilised in ddH.sub.2O with the aid of a water bath sonicator. The construct is frozen at −20° C., and thawed prior to gavage.

(22) Protocol

(23) Non-recovery anaesthesia is induced with inhalation of isoflurane followed by IP injection of sodium pentobarbital. An IV cannula is inserted (maintenance doses of anaesthesia are administered through IV cannula). A tracheotomy is performed and a feeding tube inserted, which is followed by a 2-hour recovery period.

(24) The construct is administered by gavage (in 5 ml ddH.sub.2O) via feeding tube followed by a 1-hour recovery period.

(25) Perfusion fixation is carried out (4% PFA), brains removed and incubated in post-fix solution then 30% sucrose solution. The brains are flash frozen and sectioned at 44 μm on a freezing microtome. The free-floating sections are subjected to fos immunohistochemistry.

(26) The same protocol is followed for control animals, except that no substance is gavaged.

(27) Fos+ Nuclei Counts

(28) The results show a significant increase in Fos+ nuclei/section in the paraventricular nucleus of rats administered the construct when compared to control rats.

Example 6

(29) Treatment of Oestrus-Associated Behaviour

(30) Three adult mares with behavioural problems associated with ovulation were administered 5 g of the construct with their evening feed on the day that it was evident that they were in the oestrus phase of the oestrous cycle.

(31) A veterinary-gauged semi-quantitative clinical scoring system was adopted to assess behaviour: 1=normal good behaviour; 2=mildly bad behaviour; 3=moderately bad behaviour; 4=severely bad behaviour; and 5=very severe bad behaviour with significant and often dangerous handling/management problems.

(32) Bad behaviour was characterised by a wide range of clinical signs, including squirting, kicking, biting, squealing, aggression, grumpiness, bucking, being easily spooked, anxiety, neighing when ridden, wind sucking, weaving, frequent urination, difficulty catching, tacking-up, loading and riding outside the stables, and poor performance.

(33) Prior to administration of the construct the behavioural scores were 4.5 indicating behaviour creating severe management problems. The next morning all three animals were behaving normally with behavioural scores of 1.5, thus demonstrating a stark and reproducible improvement in behaviour following administration of a construct of the invention.

Example 7

(34) Modulation of Pituitary Gland Activity

(35) A human subject is administered 1 g of the construct formulated as part of a yogurt product. Blood samples are taken prior to administration of the construct and 1, 2, 5, and 10 hours after administration, plasma is isolated and subjected to mass spectrometry. The mass spectrometry data show increased secretion of pituitary gland hormones, including oxytocin and ACTH, associated with administration of the construct.

Example 8

(36) Treatment of Rheumatoid Arthritis

(37) A patient diagnosed with rheumatoid arthritis is administered with 1 g of the construct daily, and a change in symptoms (such as: joint pain and swelling; stiffness; fatigue; depression; irritability; anaemia; and flu-like symptoms) monitored. After 1 week of treatment the symptoms are vastly reduced/eliminated, in particular, the patient has improved mobility and reduced pain.

Example 9

(38) Treatment of Periodontal Disorders

(39) A construct manufactured as per Example 1 was formulated as a toothpaste for application to the teeth and gums. The toothpaste was prepared by the addition of rapeseed oil to a finely ground powdered form of the construct with stirring at 22° C. The rapeseed oil was added in excess until a paste of appropriate density was formed. The paste was then loaded into flexible tubes which were subsequently sealed.

(40) To assess gum health, pocket depth was measured using a probe in accordance with routine dental hygiene techniques. Two human subjects were instructed to brush once daily in the evening either instead of using standard toothpaste or after such use with vigorous washing to remove any residual standard toothpaste for 2 minutes with the toothpaste formulation comprising the construct for a period of at least 4 weeks. Pocket depth was measured before and after the period of 4 weeks, and results are presented in the table below:

(41) TABLE-US-00003 Pre-Treatment Worst Post-Treatment Subject Pocket Depth (mm) Pocket Depth (mm) 1 5 0 2 8 2

(42) Hence, it was concluded that the toothpaste comprising the construct reduced the pocket depth, thus improving gum health and providing a treatment for periodontal disorders.

Example 10

(43) Largescale Periodontal Treatment Trial

(44) Twenty patients in Sydney, Australia and 10 patients in Szeged, Hungary, are selected for participation in this trial. Full periodontal charting is carried out for each patient prior to treatment to indicate the state of health of the bone and gums around each tooth. The patients are given instructions on how to use the toothpaste of Example 9, and after a period of time (a maximum of 30 days) their gum health is re-examined. The examination consists of the measurement of pocket depth, the measurement of the cemento enamel junction to the gingival margin and bleeding of the gums with probing, each of which is improved following use of the toothpaste of the invention.

Example 11

(45) Treatment of Bowel Inflammation

(46) An experiment is set up to explore the inflammation in the bowel of rats post introduction of trinitrobenzenesulphonic acid transanally to the colon. This agent causes an acute inflammation peaking at three days after introduction of the irritant. Tissue levels of tumour necrosis factor, the histopathological changes, and the gut flora at the site of inflammation are measured. These parameters of response are assessed in rats given ten day in advance feed containing the construct of the invention and a control feed with no product incorporated.

(47) Experimental Procedure

(48) Male rats are used: ˜240 g (n=10-12/group).

(49) Day 0—Colonic instillation of trinitrobenzene sulphonic acid (TNBS; 10 mg).

(50) Day 3—Removal of distal colon (8 cm), opened longitudinally, washed.

(51) Evaluation of colonic damage using stereomicroscope and computerized planimetry.

(52) Determination of myeloperoxidase activity and other biomarkers including TNF.

(53) Primary Endpoints:

(54) Area of Colonic Damage.

(55) Macroscopic Colitis Damage Score.

(56) Myeloperoxidase activity (MPO-index of neutrophil infiltration).

(57) Food weights (commencing Day—11 i.e. 24 h prior to challenge).

(58) Measurement of colonic tissue TNF-alpha (for all animals in all groups).

(59) Measurement of colonic tissue IL-B (for all animals in all groups).

(60) Secondary Endpoints:

(61) Colonic segment weight (index of tissue oedema).

(62) Daily body weight.

(63) The feed containing the construct of the invention is found to reduce the induced inflammation and all parameters thereof.

Example 12

(64) Modulation of Equine Microbiota

(65) Phase 1

(66) In vitro analysis is performed of the effects of direct application of a construct of the invention to metabolic parameters of equine gut microbiota including gas production and production of low weight fatty acids with the necessary controls.

(67) Phase 2

(68) The array of bacteria in the gut microbiota of ten horses is analysed twice during a period of a fortnight followed by administration of feed comprising a construct of the invention or a control feed lacking said construct. Weekly observations on the gut microbiota during the following three weeks is performed.

(69) The results demonstrate that a construct of the invention has a significant effect on the gut microbiota of the fed animals, and thus supports the classification of the construct as a prebiotic.

Example 13

(70) Anxiolytic Properties of the Constructs

(71) Study 1

(72) A study is performed on horses confined in a university hospital setting prior to their confinement for the induction of anaesthesia, which is a stressor for horses. Both behavioural and cardiac rate measurements are taken for horses administered the construct and those administered a control (lacking the construct).

(73) The results demonstrate that the construct has a significant anxiolytic effect.

(74) Study 2

(75) A study is performed using horses that have been discharged from a university hospital with the recommendation of box rest. Such confinement is problematic, as it causes anxiety for the horses, leading to owner non-compliance of this veterinary recommendation. Behavioural and cardiac measurements are taken for horses administered the construct and those administered a control (lacking the construct).

(76) The results demonstrate that the construct calms the horses during the confinement period, leading to improved owner compliance with regard to the box rest.

Example 14

(77) Treatment of Oral Disorders

(78) The toothpaste manufactured in accordance with Example 9 was provided to two dental practices for the evaluation of efficacy.

(79) One of the dental practices that works solely with implants gave the paste to patients after putting in the implants and reported better healing rates, as measured by the dentist.

(80) In another practice, patients suffering from chronic gingivitis showed significant improvements, in that their gum pockets started to return to normal. Moreover, two elderly women suffering from chronic gingivitis also showed significant improvement in their gum health.

(81) All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.